Tuberculosis (TB) remains a major threat to global public health and infection with Mycobacterium tuberculosis (Mtb) is estimated to result in over 2 million deaths annually. Current TB treatments are long and arduous and contribute to the emergence of multi-drug resistant (MDR)- and extensively drug-resistant (XDR)-TB. In order to develop new vaccines and drugs for TB, we need to understand how Mtb evades innate immunity and survives in hostile immune environments. A central feature of Mtb pathogenesis is its ability to grow within macrophages, modulate their activities and interfere with microbicidal functions. We have identified a cell envelope-localized lipoprotein encoding a serine hydrolase, Rv2224c, which is critical for Mtb virulence in vivo, survival in macrophages and resistance to cell envelope-directed stresses. We have named this protein Hip1 (Hydrolase important for pathogenesis 1). We demonstrate that Hip1 suppresses innate immune responses; hip1 mutants induce enhanced proinflammatory responses downstream of Toll-like receptor (TLR)-signaling in macrophages. We hypothesize that Hip1 modifies cell envelope or secreted substrates to modulate innate immunity and host defense. The focus of this application is to understand how Hip1 suppresses host innate immune pathways and determine the molecular and biochemical mechanisms for Hip1 function. The specific aims are (1) To study the role of Hip1 in suppressing host responses. We will define the TLR-dependent pathways modulated by Hip1 and evaluate whether Hip1-mediated immune suppression promotes Mtb growth in macrophages. We will also determine whether enhanced innate immune responses elicited by the hip1 mutant impact adaptive immunity by studying the priming and functionality of T cell responses. (2) To determine the molecular basis for Hip1 function. First we will characterize the molecular and biochemical basis for Hip1 interaction with a candidate physiological substrate, GroEL2. We will investigate the functional significance of Hip1-dependent cleavage of GroEL2 and its contribution to immune suppression and resisting antimicrobial defense. Second, we will use a panel of synthetic substrates to evaluate the range and specificity of Hip1 hydrolase activity. Finally we will identify additional Hip1 interaction partners and physiological substrates using genetic and biochemical approaches.